C57BL/6 Mice Are Resistant to Acute Restraint Modulation of Cutaneous Hypersensitivity

62, 250 –256 (2001) Copyright © 2001 by the Society of Toxicology TOXICOLOGICAL SCIENCES C57BL/6 Mice Are Resistant to Acute Restraint Modulation of Cutaneous Hypersensitivity Melanie S. Flint and Sally S. Tinkle 1 Toxicology and Molecular Biology Branch, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, 1095 Willowdale Road, MS 3014, Morgantown, West Virginia 26505 Received February 19, 2001; accepted May 3, 2001 The skin, due to its external anatomical location, represents a unique immunological interface for interactions with occupational and environmental exposures and also provides an organ system in which to evaluate the interplay between the central nervous system and the peripheral immune response. Over recent years, evidence has accumulated to suggest that stress plays an important role in several types of chronic health problems including modulation of immunologic diseases 1 To whom correspondence should be addressed. Fax: (304) 285-5708. E-mail: . (Dobbs et al., 1993; Kodama et al., 1999; Zhang et al., 1998). These data suggest that combined exposure to a workplace stressor and a sensitizing chemical may alter the time course and magnitude of the allergic response; however, the molecular interaction between the stress response and the cutaneous immune response is incompletely understood. Psychological stressors activate the hypothalamic pituitary adrenal axis (HPAA), which in turn, stimulates the release of glucocorticoids and catecholamines from the adrenal gland (Tomaszewska and Przekop, 1997). Glucocorticoids are major mediators of the stress response and modulate many signaling events in the immune response. Previous studies have shown that inflammatory products, such as the cytokines IL-1 and tumor necrosis factor-␣ (TNF-␣), can activate the HPAA and stimulate the release of neurohormones. Glucocorticoids, such as corticosterone, modulate antigen presentation, cytokine production, T-cell expansion, and natural killer cell activity (Belsito et al., 1982; Bonneau et al., 1997; Chrousos and Gold, 1992; Maes et al., 1998; Snyder and Unanue, 1982; Steer et al., 1998; Wiegers and Reul, 1998). Acute restraint of mice, which has many physiological similarities to emotional stress in humans, is often used to examine the influence of stress on the murine immune system (Sheridan et al., 1994; Dhabhar et al., 1997, 1999; 2000, 2001; Zhang et al., 2000). However, different strains of rodents respond differently to the effects of restraint (Dhabhar et al., 1995a,b; Sternberg et al., 1992). BALB/c mice appear to be highly responsive to stressors. They produce high levels of plasma adrenocorticotropin releasing hormone (ACTH) and corticotropin releasing hormone (CRH), and they exhibit more anxiety, as determined by behavioral disturbances than do DBA, C3H, and C57 mice (Anisman et al., 1998; Thompson, 1953). Shanks et al. (1990) demonstrated similar basal corticosterone levels in 6 strains of mice; however, in response to a stressor, the magnitude and the rate of clearance of corticosterone differed significantly between the strains. C57BL/6 mice have been reported to be relatively stress resistant and although they have basal corticosterone levels similar to BALB/c mice, they produce lower concentrations of ACTH in response to acute stressors (Anisman et al., 1998; Shanks et al., 1990). We have 250 C57BL/6 mice, in contrast to BALB/c mice, display minimal behavioral changes in response to environmental stressors and are considered relatively stress-resistant. We have shown that application of acute restraint prior to chemical challenge enhanced cutaneous hypersensitivity (CHS) in BALB/c mice and that this enhanced response is partially glucocorticoid dependent. Due to strain differences in the immune response and in the response to environmental stressors, we hypothesized that acute restraint would not enhance CHS in the less stress-sensitive C57BL/6 mice. We sensitized and challenged C57BL/6 mice with the contact sensitizer, 2, 4-dinitrofluorobenzene (DNFB) in the presence and absence of restraint. Acute restraint, applied prior to chemical challenge, significantly increased serum corticosterone, but to concentrations approximately 60% of those reported for BALB/c mice. Neither restraint nor the exogenous administration of corticosterone enhanced chemical-induced ear swelling in C57BL/6 mice. Pharmacological interruption of the hypothalamic pituitary adrenal axis (HPAA) with the glucocorticoid type II receptor antagonist, RU486, did not alter the development of CHS, however, adrenalectomized (ADX) mice exhibited decreased ear swelling, a measurement that was decreased further by restraint. Combined application of acute restraint and corticosterone prior to chemical challenge significantly enhanced the ear swelling response in C57BL/6 wild-type mice. These data confirm that C57BL/6 mice have a blunted corticosterone response to restraint and that acute restraint does not modulate cutaneous hypersensitivity. Furthermore, our data demonstrate that stress-resistance is not conferred exclusively through the glucocorticoid pathways. Key Words: stress; corticosterone; RU486; skin; sensitization. 251 RESTRAINT STRESS AND C57BL/6 MICE MATERIALS AND METHODS Mice. Young adult male C57BL/6 mice weighing 20 –25 g were purchased from Jackson Laboratories (Bar Harbor, ME). All animal protocols were approved by the NIOSH Animal Care and Use Committee. The animal room was maintained on a 12-h light/dark cycle, and lights went on at 0600 h and off at 1800 h. All animals were given food and tap water ad libitum according to ALAC approved guidelines. Adrenalectomized (ADX) C57BL/6 mice received 30 ␮g/ml of corticosterone in their drinking water during transportation and were housed individually. Upon arrival, ADX mice were rested for 5 days following removal of corticosterone from the water and administered 0.9% saline and sucrose in their drinking water throughout the course of the experiment. Induction of allergic contact dermatitis (ACD). Prior to the experiment, animals were weighed, numbered, and shaved on the back. On days 1 and 2 of the experiment 100 ␮l of 0.5% 2, 4-dinitrofluorobenzene (DNFB; SigmaAldrich, St. Louis, MO), diluted in a vehicle of 4:1 acetone:olive oil (AOO) was applied slowly to the back skin with a micropipette. On day 5, baseline ear thickness was measured for the right and left pinnae. On day 6, the right pinnae were challenged with 50 ␮l of 0.25% DNFB and the left pinnae were treated with AOO. For application of restraint prior to challenge, mice were restrained for 2 h on day 6. The thickness of the right and left ear pinnae were measured 24, 48, and 72 h after challenge using a digital micrometer. Manipulation of the HPAA Restraint. Each mouse was placed in an adequately ventilated 50 ml conical plastic tube (Corning Inc., Corning, NY) for 15 min to 2 h as specified in each experiment. Mice wer (...truncated)